Measurement of DNA Repair Proteins in Human Tissues using LC-MS/MS

1. National Institute of Standards and Technology Gaithersburg, Maryland, USA Measurement of DNA repair proteins in human tissues by liquid chromatography-tandem mass spectrometry with isotope-dilution Miral Dizdaroglu

2. Sites of oxidatively induced damage in DNA OH, eaq―, H DNA base damage DNA sugar damage 8,5'-cyclopurine-2'-deoxynucleosides Tandem lesions Clustered sites DNA-protein cross-links Single- and double-strand breaks Abasic sites OH Reviewed in: Dizdaroglu, M. and Jaruga, P., Free Radic. Res.46, 382-419, 2012

3. Products of oxidatively induced damage to DNA bases guanine-derived products (5'R)-8,5'-cyclo-2'-deoxyguanosine (5'S)-8,5'-cyclo-2'-deoxyguanosine adenine-derived products (5'R)-8,5'-cyclo-2'-deoxyadenosine (5'S)-8,5'-cyclo-2'-deoxyadenosine cytosine-derived products thymine-derived products Reviewed in: Dizdaroglu, M. and Jaruga, P., Free Radic. Res.46, 382-419, 2012

4. DNA repair in cancer therapy One important mechanism by which cancer cells can develop resistance to therapy is to increase their DNA repair capacity. The efficacy of anticancer drugs and radiation can be reduced in cancer cells by increased DNA repairthat remove DNA lesions before they become toxic. Inhibition of DNA repair DNA repair pathways are promising targets for novel cancer treatments Reviewed in: Madhusudan, S. and Middleton, M. R., Cancer Treatment Reviews 31, 603-617, 2005. Helleday, T., Petermann, E., Lunding, C., Hodgson, B. and Sharma, R.A., Nature Reviews Cancer 8, 193-204, 2008 Helleday, T., European J. Cancer 44, 921-927, 2008 Raffoul, J.J., Heydari, S.R. and Hillman, G.G., Journal of Oncology , 2012 Kelley, M., DNA Repair in Cancer Therapy, Elsevier, 2012

5. Targeted DNA repair proteins in base excision repair pathway Poly(ADP-ribose) polymerase 1 (PARP1) PARP1 is required for the efficient repair of AP sites and single-stranded DNA breaks. Cells deficient in BRCA1 or BRCA2 are highly sensitive to PARP1 inhibition. Apurinic/apyrimidic endonuclease 1 (APE1) APE1 hydrolyzes the phosphate bond at 5' to AP site, causing a strand breaks and leaving a 3'-OH group and a 5‘-deoxyribose-phosphate terminus. DNA glycosylases NEIL1, NEIL2, NEIL3, OGG1, NTH1 MTH1 MTH1 dephosporylates modified 2‘-deoxynucleoside triphosphates in the nucleotide pool to prevent incorporation of DNA lesions during DNA replication. Reviewed in: Helleday, T., Petermann, E., Lunding, C., Hodgson, B. and Sharma, R.A., Nature Reviews Cancer 8, 193-204, 2008 Wilson III, D.M. and Simeonov, A., Cell. Mol. Life Sci. 67, 3621-3631, 2010 Kelley, M., DNA Repair in Cancer Therapy, Elsevier, 2012 Gad, H., et al. Nature 508, 215-221, 2014

6. DNA repair in cancer therapy “A knowledge of DNA repair proteins’ overexpression or underexpression in cancers will help predict and guide development of treatments, and yield the greatest therapeutic response.” Kelley, M. R., DNA Repair in Cancer Therapy, Elsevier, 2012 To use DNA repair proteins as disease biomarkers or to determine the DNA repair capacity in tissues, the measurement of the levels of DNA repair proteins in vivo will be necessary. Mass spectrometric techniques with isotope-dilution will be the techniques of choice for accurate measurement of DNA repair proteins in tissues.

7. Apurinic/apyrimidinic endonuclease 1 (APE1) DNA repair activity of APE1 is critical for cell viability. 1. Complete absence of APE1 is associated with embryonic lethality in mice 2. APE1 depletion hypersensitizes cells to DNA damage 3. Overexpression of APE1 protects cells from DNA damage APE1 expression is increased in human cancers 1. Nuclear and/or cytoplasmic overexpression of APE1 occurs in breast, cervical, colon, head & neck, lung, melanoma, ovarian, prostate, etc., cancers 2. Increased APE1 expression is associated with resistance to chemo- and radiation therapies APE1 as a predictive and prognostic biomarker 1. Alterations in APE expression levels and subcellular localization may have predictive and prognostic significance in many human cancers 2. Nuclear localization is associated with good prognostic features 3. Cytoplasmic localization is associated with poor survival outcomes Positive identification and accurate quantification of APE1 in tissues is essential for its use as an efficient biomarker Reviewed in: Friedberg, E. C., Walker, G. C., Siede, W., Wood, R. D., Schultz, R. A. and Ellenberger, T., DNA Repair and Mutagenesis, 2006 Abbotts, R. and Madhusudan, S., Cancer Treat Rev. 36, 425-435, 2010

8. DNA-damaging agent APE1 repaired DNA DNA repair activity of APE1 Approximately 10000 AP sites are formed per day per cell spontaneous hydrolysis AP site APE1 APE1 hydrolyzes the O-P bond 5' to the AP site, yielding 2'-deoxyribose-5'-phosphate and 3'-OH end base excision repair pathway Reviewed in: Friedberg, E. C., Walker, G. C., Siede, W., Wood, R. D., Schultz, R. A. and Ellenberger, T., DNA Repair and Mutagenesis, 2006 excision of a modified base

9. Production, isolation and purification of 15N-hAPE1 protein markers (kDa) protein markers (kDa) 15N-hAPE1 hAPE1 15N-hAPE1 1 2 3 4 5 6 175 250 150 80 100 75 58 46 50 37 30 35.532 kDa 35.970 kDa 25 25 SDS-PAGE analysis of hAPE1 and 15N-hAPE1 20 Lane 1: Uninduced cell extract Lane 2: Induced cell extract Lane 3: 70,000 x g supernatant fraction Lane 4: Flow through from DEAE cellulose column Lane 5: Flow through from CM cellulose column Lane 6: Purified 15N-hAPE1 Kirkali, G., Jaruga, P., Reddy, P. T., Tona, A., Nelson, B. C., Li, M., Wilson III, D. M. and Dizdaroglu, M., PLOS ONE 8 (7), e69894, 2013

10. Measurement of molecular masses of hAPE1 and 15N-hAPE1 by Orbitrap mass spectrometry hAPE1 mass-to-charge (m/z) 15N-hAPE1 mass-to-charge (m/z) Kirkali, G., Jaruga, P., Reddy, P. T., Tona, A., Nelson, B. C., Li, M., Wilson III, D. M. and Dizdaroglu, M., PLOS ONE 8 (7), e69894, 2013

11. Calculation of fragment masses of tryptic peptides using NIST Mass and Fragment Calculator

12. 11 45e7 12 8 3 35e7 10 4 7 2 1 intensity 25e7 5 14 6 9 15e7 13 5e7 2 4 6 8 10 12 14 16 time (min) 11 100e7 80e7 8 3 10 12 intensity 60e7 5 7 2 1 4 40e7 14 6 9 13 20e7 2 4 6 8 10 12 14 16 time (min) Measurement of human APE1 by LC-MS/MS with isotope-dilution Sequence of hAPE1 Total-ion-current profiles of tryptic peptides hAPE1 mpkrgkkgavaedgdelrtepeak k sktaak k ndkeaagegpalyedppdqktspsgkpatlkicswnvdglrawik k kgldwvkeeapdilclqetkcsenklpaelqelpglshqywsapsdkegysgvgllsrqcplkvsygigdeehdqegrvivaefdsfvlvtayvpnagrglvrleyrqrwdeafr k flkglasrkplvlcgdlnvaheeidlrnpkgnk k nagftpqerqgfgellqavpladsfrhlypntpyaytfwtymmnarsknvgwrldyfllshsllpalcdskirsk algsdhcpitlylal318 Molecular mass 35.5 kDa Identified peptides 15N-hAPE1 Kirkali, G., Jaruga, P., Reddy, P. T., Tona, A., Nelson, B. C., Li, M., Wilson III, D. M. and Dizdaroglu, M., PLOS ONE 8 (7), e69894, 2013

13. Full scan-mass spectrum of a tryptic peptide of hAPE1 and its 15N-labeled analog 100 (M+2H)2+ 652.1 90 80 70 VQEGETIEDGAR 60 Relative Abundance (%) 50 659.9 40 30 MH+ 20 1303.1 10 0 600 700 800 900 1000 1100 1200 1300 1400 mass-to-charge (m/z) 100 (M+2H)2+ 659.9 1319.1 90 80 15N-VQEGETIEDGAR 70 60 Relative Abundance (%) 50 40 30 MH+ 20 1319.1 Kirkali, G., Jaruga, P., Reddy, P. T., Tona, A., Nelson, B. C., Li, M., Wilson III, D. M. and Dizdaroglu, M., PLOS ONE 8 (7), e69894, 2013 10 0 600 700 800 900 1000 1100 1200 1300 1400 mass-to-charge (m/z)

14. Product ion spectra of a tryptic peptide of hAPE1 and its 15N-labeled analog MH+m/z 1848.0 (M + 2H)2+ m/z 924.5 b3 b4 b7 b3 b4 b7 15N-Q–G–F–G–E–L–L–Q–A–V–P–L–A–D–S–F–R Q–G–F–G–E–L–L–Q–A–V–P–L–A–D–S–F–R Mass transitions m/z 924.5 → m/z 805.4 m/z 924.5 → m/z 904.5 m/z 924.5 → m/z 975.5 m/z 924.5 → m/z 1103.6 m/z 924.5 → m/z 1216.7 m/z 924.5 → m/z 1329.8 m/z 924.5 → m/z 1458.8 y13 y12 y11 y10 y9 y8 y7 y6 y5 y3 y13 y12 y11 y10 y9 y8 y7 y6 y5 y3 y7 805.1 100 90 80 70 y9 a9 b6 –H2O 60 975.4 y3 y8 915.7 614.4 relative abundance (%) y10 –NH3 50 409.2 b5 –H2O y11 904.4 b2 y10 1087.1 501.14 40 b3 1216.6 b7 1103.5 186.0 y5 y6 333.1 745.3 30 y12 595.3 y13 708.3 1329.8 20 1458.8 10 MH+m/z 1870.0 (M + 2H)2+ m/z935.5 0 200 400 600 800 1000 1200 1400 mass-to-charge (m/z) Mass transitions m/z 935.5 → m/z 815.4 m/z 935.5 → m/z 915.5 m/z 935.5 → m/z 987.5 m/z 935.5 → m/z 1117.6 m/z 935.5 → m/z 1231.7 m/z 935.5 → m/z 1345.8 m/z 935.5 → m/z 1475.8 y7 815.2 100 90 80 70 b5 –H2O a9 b6 –H2O 506.6 y3 b3 60 y9 y11 925.93 y5 620.0 415.14 b2 relative abundance (%) 336.6 987.5 50 y10 1231.3 603.04 189.1 40 b7 y8 1117.4 y12 y6 753.4 915.4 30 1345.9 y10 –NH3 717.4 20 1099.4 Kirkali, G., Jaruga, P., Reddy, P. T., Tona, A., Nelson, B. C., Li, M., Wilson III, D. M. and Dizdaroglu, M., PLOS ONE 8 (7), e69894, 2013 y13 1476.0 10 0 200 400 600 800 1000 1200 1400 mass-to-charge (m/z)

15. Determination of optimal collision energies M. Kinter and N.E. Sherman, Protein Sequencing and Identification Using Tandem Mass Spectrometry, Wiley, 2000 Reddy, P.T., Jaruga, P., Kirkali, G., Tuna, G., Nelson, B.C. and Dizdaroglu, M., J. Proteome Res. 12, 1049-1061, 2013

16. Enrichment of APE1 by HPLC from protein extracts of human cells 500 450 nuclear extract MCF-7 cells 400 350 Absorbance (220 nm) 300 250 200 150 100 10 12 14 16 18 20 22 24 26 Time (min) cytoplasmic extract MCF-7 cells 600 500 Absorbance (220 nm) 400 300 200 hAPE1 hAPE1 100 0 10 12 14 16 18 20 22 24 26 Kirkali, G., Jaruga, P., Reddy, P. T., Tona, A., Nelson, B. C., Li, M., Wilson III, D. M. and Dizdaroglu, M., PLOS ONE 8 (7), e69894, 2013 Time (min)

17. Identification and quantification of APE1 in human MCF-10A cells 7.36 WDEAFR 15000 m/z 412.2 → m/z 637.3 5000 0 7.37 15N-WDEAFR 20000 m/z 417.2 → m/z 645.3 NAGFTPQER 4.84 10000 20000 m/z 510.2 → m/z 834.4 0 8.27 10000 GLDWVK 40000 0 m/z 359.2 → m/z 547.3 4.84 15N-NAGFTPQER 20000 20000 m/z 517.2 → m/z 845.4 0 15N-GLDWVK 8.27 10000 100000 0 m/z 363.2 → m/z 553.3 5.14 50000 NVGWR 0 50000 Intensity m/z 316.2 → m/z 418.2 8.80 EGYSGVGLLSR 40000 0 m/z 569.3 → m/z 545.3 5.13 15N-NVGWR 20000 0 50000 m/z 321.2 → m/z 425.2 8.80 15N-EGYSGVGLLSR 30000 0 Intensity m/z 576.3 → m/z 553.3 5.30 GAVAEDGDELR 10000 15000 0 m/z 566.3 → m/z 904.4 14.70 QGFGELLQAVPLADSFR 3000 5000 0 m/z 924.5 → m/z 805.4 5.30 15N-GAVAEDGDELR 1000 20000 0 m/z 573.3 → m/z 915.4 14.69 10000 15N-QGFGELLQAVPLADSFR 4000 0 m/z 935.5 → m/z 815.4 7.06 EAAGEGPALYEDPPDQK 2000 3000 0 m/z 893.9 → m/z 584.3 0 2 4 6 8 10 12 14 16 1000 Time (min) 0 7.04 15N-EAAGEGPALYEDPPDQK 10000 m/z 903.4 → m/z 591.3 5000 0 0 2 4 6 8 10 12 14 16 Time (min) Kirkali, G., Jaruga, P., Reddy, P. T., Tona, A., Nelson, B. C., Li, M., Wilson III, D. M. and Dizdaroglu, M., PLOS ONE 8 (7), e69894, 2013

18. Identification and quantification of APE1 in human cultured cells and mouse liver Levels of hAPE1 in human normal and cancer cell lines Levels of hAPE1 in mouse liver p 0.0001 p 0.0001 MCF-7 hAPE1 level (ng/μg protein) p 0.0001 p 0.0001 p 0.0015 p 0.0001 HepG-2 hAPE1 level (ng/μg protein) MCF-10A MCF-10A: mammary gland epithelial cell line MCF-7: mammary gland epithelial adenocarcinoma cell line HepG-2: hepatocellular carcinoma cell line Kirkali, G., Jaruga, P., Reddy, P. T., Tona, A., Nelson, B. C., Li, M., Wilson III, D. M. and Dizdaroglu, M., PLOS ONE 8 (7), e69894, 2013

19. Identification and quantification of APE1 in human breast tissues disease-free breast tissue malignant breast tissue Levels of hMTH1 in human disease-free breast tissues and malignant breast tumors p 0.0001 Unpublished results

20. MTH1 nucleotidase 8-OH-dGTP 8-OH-dGMP 8-OH-dG hydrolysis Sanitation of the nucleotide pool MTH1 hydrolyzes oxidized 2’-deoxynucleoside triphosphates to monophosphates in the nucleotide pool. As a result, DNA polymerases cannot insert the wrong base across from the normal base, maintaining transcription fidelity, thus inhibiting mutagenesis. 8-OH-dGMP cannot be rephosphorylated by guanylate kinase, which phosphorylates dGMP. Reviewed in: Friedberg, E. C., Walker, G. C., Siede, W., Wood, R. D., Schultz, R. A. and Ellenberger, T., DNA Repair and Mutagenesis, 2006

21. Inhibition of MTH1 in cancer therapy Nature, 508, 215-221, 2014

22. Production, isolation and purification of 15N-hNTH1 Separation of hMTH1 and 15N-hMTH1 by HPLC. The elution profiles were superimposed. SDS-PAGE analysis of hMTH1 and 15N-hMTH1 Coskun, E., Jaruga, P., Jemth, A.-S., Loseva, O., Scanlan, L.D., Tona, A., Lowenthal, M.S., Helleday, T. and Dizdaroglu, M., DNA Repair ( in press).

23. Measurement of the masses of hMTH1 and 15N-hMTH1 by QToF LC/MS Coskun, E., Jaruga, P., Jemth, A.-S., Loseva, O., Scanlan, L.D., Tona, A., Lowenthal, M.S., Helleday, T. and Dizdaroglu, M., DNA Repair ( in press).

24. Measurement of human MTH1 by LC-MS/MS with isotope-dilution Total-ion-current profiles of tryptic peptides Sequence of hMTH1 p18 isoform hMTH1 156 molecular mass 17.95 kDa Sequences of the identified peptides 15N-hMTH1 Coskun, E., Jaruga, P., Jemth, A.-S., Loseva, O., Scanlan, L.D., Tona, A., Lowenthal, M.S., Helleday, T. and Dizdaroglu, M., DNA Repair ( in press).

25. Determination of optimal collision energies Coskun, E., Jaruga, P., Jemth, A.-S., Loseva, O., Scanlan, L.D., Tona, A., Lowenthal, M.S., Helleday, T. and Dizdaroglu, M., DNA Repair ( in press).

26. Identification and quantification of MTH1 in human cultured cells MCF-10A cells MCF-7 cells p 0.0001 Levels of hMTH1 in human normal and cancer cell lines p 0.0001 MCF-10A: mammary gland epithelial cell line MCF-7: mammary gland epithelial adenocarcinoma cell line HeLa: cervix epithelial adenocarcinoma cell line HepG-2: hepatocellular carcinoma cell line Coskun, E., Jaruga, P., Jemth, A.-S., Loseva, O., Scanlan, L.D., Tona, A., Lowenthal, M.S., Helleday, T. and Dizdaroglu, M., DNA Repair ( in press).

27. Identification and quantification of MTH1 in human breast tissues A: disease-free breast tissues B: malignant breast tumors Levels of hMTH1 in human disease-free breast tissues and malignant breast tumors p 0.0001 Coskun, E., Jaruga, P., Jemth, A.-S., Loseva, O., Scanlan, L.D., Tona, A., Lowenthal, M.S., Helleday, T. and Dizdaroglu, M., DNA Repair ( in press).

28. Conclusions Oxidative stress caused in vivo by endogenous and exogenous DNA-damaging agents leads to the formation of a plethora of lesions in DNA. DNA lesions are repaired in vivo by a variety of DNA repair mechanisms. Cancer cells resist to therapy by greater DNA repair capacity than in normal cells. DNA repair proteins are promising targets for novel cancer treatments. DNA repair inhibitors are being developed worldwide as potential drugs. Accurate measurement of DNA repair proteins’ overexpression or underexpression in cancers may help predict and guide development of treatments, and yield the greatest therapeutic response. LC-MS/MS with isotope-dilution using stable isotope-labeled analogs is well suited for the positive identification and accurate quantification of DNA repair proteins in human tissues.

29. Collaborators Pawel Jaruga, NIST Erdem Coskun, NIST Güldal Kirkali, NIST and NIH Prasad T. Reddy, NIST Bryant C. Nelson, NIST Mark S. Lowenthal, NIST Leona D. Scanlan, NIST Alex Tona, NIST Gamze Tuna, NIST Thomas Helleday, Sweden Ann-Sofie Jemth, Sweden Olga Loseva, Sweden

30. National Institute of Standards and Technology Gaithersburg, Maryland, USA Thank you